School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu 069-8501, Japan.
Environ Sci Technol. 2012 Jan 3;46(1):27-33. doi: 10.1021/es201085m. Epub 2011 Aug 3.
To assess possible impacts of environmental pollutants on gene expression profiles in a variety of organisms, we developed a novel differential display system with primer sets that are common in seven vertebrate species, based on degenerate oligonucleotide-primed PCR (DOP-PCR). An 8-mer inverse repeat motif was found in most transcripts from the seven vertebrates including fish to primates with detailed transcriptome information; more than 10,000 motifs were recognized in common in the transcripts of the seven species. Among them, we selected 275 common motifs that cover about 40-70% of transcripts throughout these species, and designed 275 DOP-PCR primers that were common to seven vertebrate species (common DOP-PCR primers). To detect genes responsive to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in developing embryos, differential display with common DOP-PCR primers was applied to embryonic liver of two avian species, the chicken (Gallus gallus) and the common cormorant (Phalacrocorax carbo), which were exposed in ovo to TCDD. The cDNA bands that showed differences between the control and TCDD-treated groups were sequenced and the mRNA expression levels were confirmed by real-time RT-PCR. This approach succeeded in isolating novel dioxin-responsive genes that include 10 coding genes in the chicken, and 1 coding gene and 1 unknown transcript in the cormorant, together with cytochrome P450 1As that have already been well established as dioxin markers. These results highlighted the usefulness of systematically designed novel differential display systems to search genes responsive to chemicals in vertebrates, including wild species, for which transcriptome information is not available.
为了评估环境污染物对各种生物基因表达谱的可能影响，我们开发了一种新的差异显示系统，该系统使用了基于简并寡核苷酸引物的 PCR（DOP-PCR）在 7 种脊椎动物中常见的引物对。在包括鱼类到灵长类动物在内的 7 种脊椎动物的大多数转录本中都发现了一个 8 个碱基反向重复基序；在 7 种物种的转录本中，有超过 10000 个基序被识别为共同存在。其中，我们选择了 275 个常见基序，这些基序覆盖了这些物种大约 40-70%的转录本，并设计了 275 个在 7 种脊椎动物中通用的 DOP-PCR 引物（通用 DOP-PCR 引物）。为了检测 2,3,7,8-四氯二苯并对二恶英（TCDD）在发育胚胎中诱导的基因，我们应用通用 DOP-PCR 引物对两种禽类，鸡（Gallus gallus）和普通鸬鹚（Phalacrocorax carbo）的胚胎肝脏进行了差异显示。在对照组和 TCDD 处理组之间显示差异的 cDNA 条带被测序，并通过实时 RT-PCR 确认了 mRNA 表达水平。这种方法成功地分离出了新的二恶英反应基因，包括鸡的 10 个编码基因、鸬鹚的 1 个编码基因和 1 个未知转录本，以及已经被证明是二恶英标志物的细胞色素 P450 1As。这些结果突出了系统设计的新型差异显示系统在搜索包括野生物种在内的脊椎动物中对化学物质有反应的基因方面的有用性，对于这些物种，转录组信息尚不可用。
